ABSTRACT The reparative capacity in human articular cartilage is generally considered to be low or negligible, and this intrinsic capacity decreases with age. As a result, articular cartilage injuries often result in irreversible damage leading to osteoarthritis (OA). We and others have recently defined heterogeneity in articular chondrocytes at both the molecular and cellular levels. Work in mice and other mammals has implicated a subset of cells in the superficial layer of articular cartilage as the source of regenerative capacity; to date, these findings have not been extended to a specific population of chondrocytes in human ontogeny. Our previous studies have shown that unlike adult chondrocytes, fetal chondrocytes are highly proliferative and migratory, and exhibit high basal levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Our preliminary data also nominate cells expressing integrin ?4 (ITGA4) and bone morphogenetic protein receptor (BMPR1B) as the most immature chondrocytes in human articular cartilage throughout human development. Moreover, we have shown at the molecular level that ITGA4+BMPR1B+ cells are enriched for active STAT3 (pSTAT3), which are known to drive proliferation, anabolism and preserve differentiation potential. Importantly, adult ITGA4+BMPR1B+ cells are localized to the superficial layer and also express the highest levels of SOX9, which is strongly identified with osteochondral progenitor identity and anabolism; indeed, ITGA4+BMPR1B+ cells are robustly chondro- and osteogenic in vitro. The percentage of ITGA4+BMPR1B+ cells and levels of pSTAT3 tightly correlate with biological age, decreasing from 20-30% in developing joints down to 1-2% in aged adult healthy cartilage. We hypothesize that active STAT3 is expressed in immature articular chondrocytes and is a permissive factor required for immature cell anabolism and differentiation in response to specific instructive signals in the niche. We propose to define the direct transcriptional targets of STAT3 in human articular chondrocytes at different ontogenic stages and under conditions similar to the pro-inflammatory state driven by IL-6 family cytokines in OA. To address how IL-6 family cytokines can drive varied biological and functional outcomes in a context- specific manner, we will employ nanoproteomics and targeted mutagenesis to determine how specific post- translational modifications in the core IL-6 family cytokine receptor gp130 differ in fetal vs. adult chondrocytes stimulated with IL-6 family cytokines. Finally, we will apply single cell RNA-Seq to further refine the molecular and cellular phenotype of immature articular chondrocytes. In parallel, we will assess the molecular and functional consequences of STAT3 gain and loss of function in articular chondrocytes. We propose that cells with higher levels of pSTAT3 will evidence broader differentiation potential in vivo, resulting from changes mediated by STAT3 in chromatin conformation. The overall impact of this highly innovative study is to define the cellular and molecular phenotype of immature articular chondrocytes throughout human ontogeny and to link this to the potential for cartilage repair and/or regeneration during aging.